The polymerase chain reaction (PCR) is a method which permits the specific in vitro amplification of DNA sequences [Mullis et al., Cold Spring Hath. Syrup. Quant. Biol., 51:263-273 (1986); Saiki et al., Science, 239:487-491 (1988); Saiki et al. , Science, 230: 1350-1354 (1985)]. PCR occurs by primer annealing to and extension from each end of a targeted sequence, and until recently has required knowledge of the primer annealing sites at each end of the targeted sequence. Recently, several methods have been developed for the amplification of unknown DNA that flanks one end of a known sequence, so that one may walk along a DNA sequence without screening a library for overlapping clones [Arnold & Hodgson, PCR Methods Applic., 1: 39-42 (1991); Collasius et al., J. Virol. Methods, 32:115-119 (1991); Edwards et al. , Nucleic Acids Res., 19: 5227-5232 (1991); Fors et al. , Nucleic Acids Res., 18: 2793-2799 (1990); Frohman et al. , Proc. Natl. Acad. Sci., 85: 8998-9002 (1988); Gibbons, et al., Proc. Natl. Acad. Sci., 88:8563-8567 (1991); Hovens and Wilks, Nucleic Acids Res., 17:4415 (1989); Jones and Winistorfer, Nucleic Acids Res., 20: 595-600 (1992); Jones and Winistorfer, PCR Methods Applic. , 2:197-203 (1993); Kandpal et al. , Nucleic Acids Res., 18:3081 (1990); Lagerstrom et al., PCR Methods Applic., 1:111-119 (1991); Loh et al. , Science, 243:2 17-220 (1989); MacGregor and Overbeek, PCR Methods Applic., 1:129-135 (1991); Mueller and Wold, Science, 246:780-786 (1989); Ochman et al. , Genetics, 120:621-623 (1988); Ohara et al. , Proc. Natl. Acad. Sci., 86:5673-5677 (1989); Parker et al., Nucleic Acids Res., 19:3055-3060 (1991); Parks et al. , Nucleic Acids Res., 19:7155-7160 (1991); Pfeifer et al., Science, 246:810-813 (1989); Riley, et al., Nucleic Acids Res., 18:2887-2890 (1990); Rosenthal and Jones, Nucleic Acids Res., 18:3095-3096 (1990); Roux and Dhanarajan, BioTechniques, 8:48-57 (1990); Shyamala and Ames, Gene, 84:1-8 (1989); Silver and Keerikatte, J. Virol. 63:1924-1928 (1989); Triglia et al., Nucleic Acids Res., 16:8186 (1988); Tormanen, et al., Nucleic Acids Res., 20:5487-5488 (1992)].
A major obstacle in using existing methods for the PCR amplification of specific sequences in genomic DNA is the occurrence of nonspecific amplification products. Under PCR conditions, the stringency of the priming [Sommer and Tautz, Nucleic Acids Res., 17:6749 (1989)] is seldom high enough to generate a pure product longer than 1 kilobase (kb) in highly complex mixtures, such as in human genomic DNA. This limits both the specificity of the reaction and the length of the amplifiable DNA. Use of nested primers [Mullis et al., Cold Spring Harbor Symposia on Quantitative Biology, Cold Spring Harbor Laboratory, L1:263-273 (1986); Haqqi et al., Nucleic Acids Res., 16:11844 (1988)] and size selection of the regions of interest by previous Southern blotting [Ochman et al., Genetics, 120:621-623 (1988); Beck and Ho, Nucleic Acids Res., 16:9051 (1988)] diminish this problem. However, high background due to insufficient stringency during the PCR amplification of genomic DNA remains a significant problem. It is not surprising, therefore, that the methods to amplify unknown flanking DNA result in limited specificity, as the initial PCR amplification using these methods does not improve upon the specificity level conferred by conventional two primer PCR. Certainly, an approach that optimizes the specificity of amplification is advantageous, regardless of the other strategies used to increase specificity (nested primers, size selection, physical separation of biotinylated products with steptavidin).
One method that has permitted the highly specific amplification of &gt;2 kb of unknown DNA that flanks a known sequence from bulk human genomic DNA is panhandle PCR [Jones and Winistorfer, Nucleic Acids Res., 20:595-600 (1992); Jones and Winistorfer, PCR Methods Applic., 2:197-203 (1993)]. This method involves a primer dependent attachment of a known sequence to the uncharacterized side of a specific DNA strand which contains an unknown sequence. This permits specific PCR amplification of the unknown DNA because known sequence now flanks the strand that contains the unknown DNA. The PCR template is generated in the following manner. First, a restriction enzyme digests DNA leaving a 5' overhang. Second, a single-stranded oligonucleotide is ligated to the restriction enzyme digested DNA resulting in the modification of the 3' end of each strand. This oligonucleotide is constructed to be complementary to the known region of DNA immediately upstream from the unknown region of DNA. Third, denaturation and self-annealing under dilute conditions results in strands of DNA, containing the complement to the ligated piece, forming a single-stranded loop, or pan portion, with a double-stranded stem of an otherwise single-stranded handle, of a panhandle structure. The sequence specific annealing that constitutes the double-stranded stem can prime template-directed DNA polymerization from the ligated oligonucleotide. This polymerization results in known DNA being placed on the uncharacterized end of the unknown DNA contained in the loop. Generation of the panhandle template permits PCR amplification of the unknown DNA because known sequence now flanks the unknown DNA in those strands that contain the unknown DNA. However, in the panhandle PCR method, the initial priming during the amplification reaction must compete with intra-strand annealing of a long inverted repeat that comprises the handle of the panhandle template, which diminishes the efficiency of this necessary first step.
The present invention provides a method that permits the highly specific amplification of &gt;2 kb of DNA that flanks the primer annealing sites from bulk human genomic DNA.